To understand the functionally significant states of a regulatory protein complex, one must directly measure the thermodynamic and kinetic forces that drive its assembly and ligand-induced conformational responses. Calmodulin (CAM), an essential eukaryotic calcium sensor, is an allosteric monomer that controls elements of neurotransmission, muscle contraction, fertility and metabolism through its calcium-dependent association with target proteins. Historically, it was viewed as having two functional states: "on" (saturated with 4 calcium ions) or "off" (apo, calcium-free). A few target proteins were recognized to reverse this logic and use the apo form of CaM as an activator. Its two homologous domains (N & C) were believed to be equivalent partners in association with target proteins. However, the two EF-hand domains of CaM are now recognized to have separable roles in activation of some targets. The major goal of this proposal is to elucidate molecular mechanisms of domain-specific transitions in CaM that govern its physiological roles. Studies by this laboratory of 2 classes of Paramecium CaM mutants, defective in regulating ion channels, demonstrated that (a) mutations in helices B & C that lower thermostability make calcium binding more favorable, (b) interactions between helices A & D are key determinants of species differences in ion binding and flexibility and (c) covalent coupling of the N- and C-domains exacerbates their differences. All of the CaM mutants studied were able to bind target peptides under both apo and calcium-saturating conditions, demonstrating that regulatory failure is occurring via altered pathways through the intermediate states (i.e., defective conformational responses or reduced binding constants). This research program will (a) determine the roles of the interdomain linker on properties of the N- and C-domain and (b) quantitatively evaluate the interactions between mutant PCaM's and selected target proteins. Conformational switching and energetics of ion and target binding will be determined using heteronuclear NMR, fluorescence, CD, mass spectroscopy, and hydrodynamic methods (ultracentrifugation, chromatography). This analysis of CaM will contribute to understanding pathways of domain interactions and the physiologically distinct roles these highly homologous domains play in target activation. This will lead to a better understanding of how synchronized changes in calcium levels modulate diverse physiological processes in eukaryotes.